The need for blood transfusions in cases of trauma is readily appreciated by most people. In a broader context, disease, deficiencies of the haematopoietic system, and insult through chemical, radiological, physical, or other means, can all have serious consequences through loss of blood, changes to the composition of blood, altered functionality of blood, and/or reduced maintenance of the circulating blood pool (homeostatic replacement and refreshment of blood and its components).
Blood is a complex biological fluid consisting of multiple cellular (e.g., erythrocytes, macrophages, lymphocytes, monocytes, platelets), and non-cellular components (e.g., plasma, immunoglobulins). In the case of cellular components, the only means through which these are currently available as a transfusable product, is through donation. Worldwide, the demand for transfusable blood products is increasing. Unfortunately this increasing demand cannot be met by the current donor system. Blood shortages are not uncommon, and loss is still a major cause of death.
Increasing stringency in donor screening, and the prevalence of blood transmissible disease such as HIV/AIDS is limiting the availability of new donors, while ageing of existing donors reduces their ability to give blood. There is also a substantial imbalance in donor availability between developed and developing/transitional regions. More than 81 million units of blood are collected each year, but only 45% of these are donated in developing or transitional countries, where greater than 80% of the world's population live.
A possible alternative to the existing blood donor system is to cultivate blood cells in vitro. By leveraging the capacity of haematopoietic stem cells to expand in number and differentiate along particular lineages in response to specific cues, it may be possible to manufacture substantial numbers of blood cells and derivatives (e.g. platelets).
In order to better understand the task of manufacturing blood cells, it is useful to understand the underlying biology of the haematopoietic system.
Human Haematopoiesis
All blood cells are derived from a common progenitor cell type known as the haematopoietic stem cell (HSC). HSC were the first human stem cell to be identified, and have been used in human therapy since the early 1950's in the form of bone marrow transplants. HSC have the ability to either self-renew, or enter a process of differentiation by which a single HSC can give rise to progeny belonging to any of the haematopoietic lineages. These lineages are broadly categorised as myeloid and lymphoid.
Cells of the myeloid lineage arise from a common myeloid progenitor and can be further categorised into erythrocytes, megakaryocytes, granulocytes, and monocytes.
Erythroid cells ultimately mature into red blood cells (erythrocytes), playing an essential role in transporting oxygen throughout the body. Megakaryocytes play a vital role in the production of cell fragments known as platelets or thrombocytes, which are essential in blood clotting. Granulocytes and monocytes are immune cells involved in both adaptive and innate immunity, with granulocytes further subdivided into neutrophils, basophils, and eosinophils. Monocytes give rise to macrophages and myeloid dendritic cells.
The lymphoid lineage consist of immune cells such B-cells, T-cells, natural killer cells, and lymphoid dendritic cells, which all play a role in adaptive immunity.
In adults, the primary site of haematopoiesis is the bone marrow. Here the HSC are believed to reside within a regulatory microenvironment or niche, which acts to maintain the HSC pool through self renewal. Progenitor cells may exit the niche and undergo a regulated process of differentiation toward mature blood cells of the lineages described previously. The HSC niche and process of differentiation are regulated by a number interacting factors. Physiochemical parameters such as dissolved oxygen and pH, the concentration of biological effectors molecules, interactions with surrounding cells, and contact with extra cellular matrix (ECM) and ECM bound factors, are all believed to a play role in the regulation HSC self-renewal and differentiation.
By identifying the specific cues required to drive differentiation towards a particular lineage, and manipulating these ex vivo in cell culture systems, it is is possible to selectively expand and differentiate HSCs into large numbers of lineage specific cells. The final cell product may be fully mature, or alternatively, a population of lineage committed, but not fully differentiated cells, can be produced.
Conditions for ex vivo production of mature blood cells from HSCs have been described, with varying degrees of success. In addition to haematopoietics, three exemplary cell types are erythrocytes, dendritic cells and megakaryocytes. A number of potential clinical uses for such ex vivo expanded haematopoietic cell populations have been proposed.
Clinically, erythrocytes, could be used to replace lost blood in cases of bleeding/trauma. Megakaryoctyes can be used to generate platelets for transfusion support in chemotherapy patients. Dendritic cells have been proposed as a means by which to train the body's immune system to recognise and attack cancer cells. Numerous other applications of ex vivo expanded blood cells are also possible, replacing or augmenting current applications for donor derived products, or representing new therapeutic avenues.
While biological cues for ex vivo expansion of haematopoietic cells have been identified, a key challenge to clinical application lies in identifying appropriate means for the large scale production of these cells. Traditional static culture systems (e.g., tissue culture flasks) cannot be readily scaled to produce clinically relevant cell numbers (e.g. at 2×1012 cells, one unit of erythrocytes would require some 5000 m2 of culture surface to produce in static flask cultures).
In order to reduce the required surface area per unit volume of culture, agitation can be used to induce mixing and hence enhance mass transfer of oxygen into the culture environment. In this way, large volume cultures can be conducted in compact geometries. Stirred bioreactor systems (or fermenters) can and have been used for the cultivation of haematopoietic cells. However, in these systems the extent of expansion achieved is poor and insufficient cell numbers are obtained. Alternative systems by which to deliver oxygen and nutrients within compact geometries, such as hollow fibre culture devices, suffer from additional engineering complications. Again taking the example of one unit of erythrocytes, some 1.5 km of fibre, providing a lumen volume of 50 L, would be required. This is far beyond the scale to which this approach has so far been successfully demonstrated.
In order to realise the potential of ex vivo expanded haematopoietic cells in a clinical setting, improved processes for their manufacture in substantial numbers are required.